The goal of this project is to develop and validate assays to detect the presence of replication competent retroviruses (RCR) in preparations of viral vectors to be used for clinical purposes. The currently recommended procedure is to test 1% of the cells and 5% of the supernatant for the presence of RCR using the S+L-assay as a readout. The S+L-assay has a number of problems, and investigators have been developing alternate assays, known as marker rescue assays, which have been reported to be more sensitivities. Because of the relative difficulty of the readout on S+L-, the first goal of this project is to validate the marker rescue lines that are currently available for distribution and to compare their sensitivity of detection to that of the S+L-. The second goal of the project is to determine the relative sensitivities of the current recommendations. Experiments have been designed to determine whether co-cultivation of vector producing cells with a permissive cell line is more sensitive than testing of supernatant from the vector producing cells alone.